1. Technical Field
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has a protein derived from a bacterium belonging to the genus Pantoea, and the protein is able to confer resistance to cysteine.
2. Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to feedback inhibition by the resulting L-amino acid (U.S. Pat. Nos. 4,346,170; 5,661,012; and 6,040,160).
A new microbial strain is disclosed which is suitable for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine which is produced from the non-enzymatic conversion of O-acetyl-L-serine, and/or thiazolidine derivatives. This new strain overexpresses at least one gene which codes for a protein that mediates cellular clearance of antibiotics or other substances that are toxic for the microorganism (EP 0885962).
A chromosomal fragment has been identified in a gene bank from Escherichia coli, which is able to increase the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids, called Orf299, was responsible. The Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. These results further indicated that ORF299 codes for an export pump responsible for excreting different metabolites of the cysteine pathway (Dassler T. et al, Mol. Microbiol.; 36(5): 1101-12 (2000).
The ORF yfiK gene was discovered to be able to increase cysteine production when overexpressed in an industrial E. coli production strain. The yfiK gene product is an integral membrane protein with about six predicted transmembrane helices, and it belongs to the RhtB family of export proteins. YfiK overproduction from a plasmid leads to drastic and parallel secretion of O-acetyl-L-serine and cysteine into the medium, but only when the organism possesses a serine transacetylase that is insensitive to feedback inhibition by cysteine. When excess O-acetyl-L-serine is added to the medium, this requirement for the presence of a feedback-insensitive serine transacetylase during cysteine secretion can be obviated both in the yfiK-carrying transformant and in the wild-type strain. A delta yfiK mutant did not show any phenotype, and was able to export O-acetyl-L-serine and cysteine when transformed with a plasmid carrying ydeD, a previously characterized, alternate O-acetyl-L-serine/cysteine exporter. Since an ydeD-yfiK double mutant showed the same pattern, it appears that YfiK and YdeD act independently. The necessity for the cell to regulate the size of the internal pool of O-acetyl-L-serine via synthesis of exporter proteins could be connected to the fact that this compound (when supplied externally) inhibits growth. Overexpression of either ydeD or yfiK alleviates this inhibition, and increases resistance to azaserine, which is an analog of O-acetyl-L-serine (Franke I et. al., J. Bacteriol.; 185(4):1161-6 (2003)).
Assembly of E. coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, the substrate of which is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, even though no single major protein was missing in the cydD periplasm. Instead, CydDC exports from the cytoplasm to the periplasm the amino acid cysteine, which can be further demonstrated by using reverted membrane vesicles that transport radiolabeled cysteine inward in an ATP-dependent, uncoupled-independent manner. New pleiotropic cydD phenotypes have been reported, including ones with sensitivity to benzylpenicillin and dithiothreitol, and ones with loss of motility. Both of these phenotypes are consistent with periplasmic defects in disulfide bond formation. The presence of exogenous cysteine was able to reverse these phenotypes and affect the levels of periplasmic c-type cytochromes in cydD and wild-type strains, but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and produced higher cytoplasmic cysteine levels, as did a mutant defective in ORF299 which encoded a transporter of the major facilitator superfamily. A cydD ORF299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. It seems likely that CydDC is responsible for the export of cysteine, which is crucial for redox homeostasis in the periplasm (Pittman M. S. et al., J Biol. Chem.; 277(51):49841-9 (2002)).
In addition to YdeD and YfiK, which have been previously reported as L-cysteine exporter proteins in E. coli, the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction was analyzed. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojlH genes reversed the growth inhibition of tnaA-disrupted E. coli cells by L-cysteine. The tnaA gene is the major cysteine desulfhydrase gene. It was found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression, which confers bicyclomycin and tetracycline resistance, specifically promotes L-cysteine export driven by the energy generated from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant produced more L-cysteine than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at substantial levels. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells (Yamada S. et al., Appl Environ Microbiol.; 72(7):4735-42 (2006)).
But currently, there have been no reports of using a bacterium having a protein derived from bacteria belonging to the genus Pantoea, and which is able to confer resistance to growth inhibition by L-cysteine in the bacterium, for the purpose of producing L-amino acids.